水中抑制物对定量PCR与膜过滤法检测E.coli的影响  

Effect of inhibitors in water on detection of Escherichia coli by quantitative PCR and membrane filtration method

作　　者：徐丽梅[1] 王晓昌[1] 周进宏[1] 吉铮[1] 张崇淼[1] 

机构地区：[1]西安建筑科技大学环境与市政工程学院西北水资源与环境生态教育部重点实验室,西安710055

出　　处：《环境工程学报》2015年第2期609-614,共6页Chinese Journal of Environmental Engineering

基　　金：国家“水体污染控制与治理”科技重大专项(2013ZX07310-001)

摘　　要：针对水中病原微生物的污染,选择大肠埃希氏菌E.coli作为病原示踪剂,以E.coli染色体上β-葡萄糖醛酸酶uid A目的基因建立了SYBR Green实时荧光定量PCR的检测方法。该定量PCR方法灵敏度高,检测限可达104CFU/L,线性关系良好,相关系数为R2=0.999。研究表明,定量PCR与菌液浓度呈显著正相关R2=0.935。通过人工投加腐殖酸、COD,研究了水中抑制物对定量PCR和膜过滤MF培养法的影响。结果显示,腐殖酸可使E.coli在培养皿上的菌落变小聚集,且显色不明显,当腐殖酸量为20 mg/L时,浓度为50 CFU/100 m L的E.coli完全受到抑制,没有菌落出现。腐殖酸对PCR扩增的抑制作用明显,当腐殖酸浓度增加到10 mg/L,定量PCR检测结果基因拷贝数减少约1 log,增加到20 mg/L定性PCR检测结果为阴性。Escherichia coli(E.coli) was selected as the pathogenic tracer.Based on the uid A gene sequence of E.coli,the SYBR Green quantitative polymerase chain reaction(q PCR) method was established.The detected limitation of q PCR was 104 CFU/L and a good linear relationship was obtained with correlation coefficient(R2) of 0.999.The concentrations of E.coli detected by q PCR and membrane filtration(MF) method were presented a significant positive correlation with R2 of 0.935.Effect of inhibitors in water on detection of E.coli by q PCR and MF method was evaluated.Results showed that the colonies gathered together and became smaller,and the color of colonies was not apparent after adding humic acid.When concentrations of E.coli and humic acid were approximately 50 CFU/100 m L and 20 mg/L,respectively,colony growth was inhibited completely on culture plate.Inhibitory effect of humic acid on q PCR detection was apparent.As humic acid concentration increased to 10 mg/L,E.coli concentration reduced approximately for 1 log.While detected result was negative when humic acid concentration was 20 mg/L.

关 键 词：E.COLI 实时荧光定量PCR 膜过滤 腐殖酸 抑制作用 

分 类 号：X832[环境科学与工程—环境工程]